To build a functional vascular network, endothelial tubes must adopt stereotyped topologies. Both multicellular and unicellular tube topologies are observed in vertebrate vasculatures. Seamless tubes are unicellular and unbound by junctions. Seamless tubes are found in multiple contexts within vertebrate vascular networks. The functional roles of seamless tubes in these contexts are poorly understood because the cellular mechanisms of seamless tube morphogenesis are poorly understood. Nevertheless, seamless tubes are conserved across phyla, including in the respiratory (tracheal) system of Drosophila melanogaster larvae, where powerful forward genetic approaches can be utilized to study seamless tube morphogenesis. To build a functional vascular network, endothelial tubes must also adopt a stereotyped shape. Endothelial cells secrete a luminal (apical) extracellular matrix (aECM) called the glycocalyx, comprised of secreted and membrane-bound glycoproteins and proteoglycans. Defects in glycocalyx structure lead to defects in endothelial tube expansion. Indeed, aECMs have a conserved role in regulating tube growth, including in Drosophila tracheal tubes. The seamless tubes of the Drosophila trachea secrete a chitin-based aECM called the cuticle, whose role in seamless tube morphogenesis not known. We have a poor understanding of molecular pathways through which individual secreted matrix factors are organized into an ordered aECM. Moreover, the molecular pathways through which aECMs shape the apical membrane to regulate tube morphogenesis are entirely unknown. I will use the seamless tubes of the Drosophila larval tracheal system as a model to elucidate novel genetic pathways controlling seamless tube shape by regulating aECM structure/function. I have identified a role for the seamless tube aECM in regulating tube shape and integrity. Chitin synthase-deficient terminal cells exhibit apical membrane cysts and discontinuities. I have also identified a set of novel mutants (ichor and asthmatic) that phenocopy chitin biogenesis mutants. I hypothesize that these mutants may affect novel pathway(s) regulating the structure or function of a seamless tube aECM. ichor and asthmatic encode zinc- finger transcription factors (CG11966 and zif, respectively), suggesting they regulate the expression or targeting of aECM components to the lumen. In Specific Aim 1, I will identify the genetic requirements for aECM organization in seamless tubes, including testing the role of a conserved polarity factor known to be downstream of Zif, in targeting aECM components to the lumen. In Specific Aim 2, I will identify cellular and molecular pathways through which the seamless tube aECM regulates tube shape. I will test a role for the seamless tube aECM in regulating apical actomyosin organization using both genetic and innovative live cell imaging techniques.